This pertains generally to compounds and to the design of these compounds targeted to bind to ribonucleic acid; and, more particularly, to compounds that bind specifically to certain nucleotide base pairs in combination with elements of the secondary structure of the minor groove of ribonucleic acid molecules.
With few exceptions, ribonucleic acid (RNA) molecules are synthesized by the transcription of specific regions of deoxyribonucleic acid (DNA). The general function of RNA is as the intermediary of protein synthesis from DNA into the amino acid sequences of protein, although RNA has recently been discovered to have other functions, including enzymatic activity.
Three principal types of RNA exist in cells; messenger RNA, transfer RNA and ribosomal RNA. The messenger RNAs (mRNA) each contain enough information from the parent DNA molecule to direct the synthesis of one more proteins. Each has attachment sites for tRNAs and rRNA. The transfer RNAs (tRNA) each recognize a specific codon of three nucleotides in a strand of mRNA, the amino acid specified by the codon, and an attachment site on a ribosome. Each tRNA is specific for a particular amino acid and functions as an adaptor molecule in protein synthesis, supplying that amino acid to be added to the distinctive polypeptide chain. Subunits of ribosomal RNA (rRNA) form components of ribosomes, the xe2x80x9cfactoriesxe2x80x9d where protein is synthesized. The subunits have attachment sites for mRNA and the polypeptide chain. The rRNAs regulate aminoacyl-tRNA binding, mRNA binding, and the binding of the initiation, elongation, and termination factors; peptide bond formation; and translocation, as reviewed by Endo, et al., J. Biol. Chem. 265:2216 (1090).
The RNAs share a common overall structure, though each kind of RNA has a unique detailed substructure. Generally, RNA is a linear, single-stranded (with a few viral exceptions), repetitive polymer in which nucleotide subunits are covalently linked to each other in sequence. Each nucleotide subunit consists of a base linked to the ribose-phosphate of the polymeric backbone. The bases in RNA are adenine (A), uracil (U), guanine (G), and cytosine (C). The sequence of bases imparts specific function to each RNA molecule. Nucleotide bases from different parts of the same or different RNA molecules recognize and noncovalently bond with each other to form base pairs. Since RNAs generally are a single covalent strand, base pairing interactions are usually intrastranded, in contrast to the interstrand base pairing of DNA. These noncovalent bonds play a major part in determining the three-dimensional structure of each of the RNAs and the interaction of RNA molecules with each other and with other molecules. The 2xe2x80x2 hydroxyl group also influences the chemical properties of RNA, imposing stereochemical constraints on the RNA structure, by restricting the ribose conformation in oligomeric RNA molecules to the C3xe2x80x2-endo conformation, in contrast to DNA, where the sugars freely interconvert between the C3xe2x80x2-endo and C2xe2x80x2-endo puckered conformations.
The RNA molecule forms a helix with major and minor grooves spiralling around the axis, as shown in FIG. 1A. Nucleotide bases are arranged near the center of the helix with the ribose phosphate backbone on the outside. The bases are planar, perpendicular to the axis, and stacked on one another. Because the helix is in the alpha form, bases and sequences of bases are most accessible from the minor groove, which is wider and more shallow than the major groove, as discussed by Arnott, et al., J. Mol. Biol. 27:525 (1967). DNA, in contrast, is found as either an A- or B-form helix, as shown in FIGS. 1A and 1B, respectively. The different types of helical structure present different molecular surfaces to the proteins with which they make sequence-specific contacts.
RNA molecules assume a greater variety of tertiary structures than do DNA molecules, because of the lack of a complementary second strand and because of the potential to form Watson-Crick intrastrand hydrogen bonds between complementary sequences which can be well separated from each other in the linear sequence. In addition, the juxtapositioning of distant bases in the sequence allows for tertiary base pairing schemes that typically are non-Watson-Crick, such as Hoogstein pairing. Consequently, in the absence of proteins, doubled stranded DNA rarely assumes the globular forms characteristic of transfer RNAs or ribosomal RNAs. The higher order DNA structures that are found in vivo, including those resulting from supercoiling and those associated with the folding of chromosomes, are dependent on topoisomerases and packaging proteins. Even so, the condensation of DNA in chromosomes results in a structure that is more rod-like than globular.
Transfer RNA is the most well characterized of the RNA molecules. One or more specific tRNAs exists for each of the twenty amino acids in cello. The tRNA molecule is a 70 to 80 nucleotide strand forming two helical regions. One helical region terminates in the anticodon loop, which base-pairs to a complementary triplet in an mRNA codon. The other helical region terminates in the amino acid acceptor helix, which recognizes and binds a specific amino acid. Base pairing, as shown by crystallographic analysis, accounts for the formation of a two-dimensional stem-loop structure similar to a cloverleaf, forming the secondary structure of the molecule. See, e.g., Holmquist, et al., J. Molec. Biol. 78:91 (1973). As tRNAs line up on the mRNA molecule, bringing their amino acids into juxtaposition, they enable conversion of sequences of nucleotides into the sequences of amino acids that form the polypeptide chains. Hou and Schimmel, Nature 333:140 (1988). This function of tRNA is essential for protein synthesis. A related function of the tRNA molecule is to activate and enable the amino acid to react with another amino acid to form the peptide bond necessary for linkage. This step is also essential for protein synthesis. During protein synthesis, xe2x80x9cthe success of decoding is crucially dependent on the accuracy of the mechanism that normally links each activated amino acid specifically to its corresponding tRNA molecules.xe2x80x9d Alberts, B., et al., Molecular Biology of the Cell, 2d ed., Garland Publishing, Inc., page 207 (1989).
The reaction in which a tRNA becomes linked to the one appropriate amino acid is catalyzed by an enzyme, aminoacyl-tRNA synthetase. Each of the twenty amino acids requires a different synthetase enzyme that recognizes it and attaches it to one of its set of cognate tRNA molecules.
Since RNA is critical to protein synthesis and the transfer of genetic information encoded in the deoxyribonucleic acid (DNA) of eukaryotic cells, bacteria, and viruses, it represents a potential mechanism by which all pathogenic agents can be inhibited. At this time, however, little progress has been made in identifying a means by which RNA can be inhibited specifically.
Drugs that are currently available act on specific biochemical pathways via interaction with a particular protein or cofactor, or by interference in general with nucleic acid synthesis or translation. Most chemotherapeutic agents act by the latter mechanism, where the most rapidly replicating cells (usually the cancer cells) are inhibited more than the slower growing cells. The compounds are toxic to all cells, however.
It is therefore an object of the present invention to provide methods to make compositions, and the products thereof, that specifically inhibit RNA.
It is another object of the present invention to provide methods for designing compounds specifically inhibiting RNA that have low toxicity to normal eukaryotic cells.
It is a further object of the present invention to provide methods for screening and administering such compounds for specific inhibition of RNA.
A method for designing compounds specifically targeting RNA sequences by modeling the compound to bind to crucial short nucleotide sequences in the RNA in combination with secondary and/or tertiary structure associated with the minor groove of the RNA. In the preferred method, a critical sequence of the RNA is identified, then computer modeling is used in combination with analysis of the targeted RNA sequence to design molecules binding to the targeted RNA by covalent or hydrogen binding. Appropriate molecules specifically inhibiting the function of the targeted RNA are synthesized using known methodology that have the required secondary structure and chemical characteristics. Molecules known to bind to RNA can also be modified using this method to increase specificity, and thereby decrease toxicity.
Much of the design of these compounds, as well as the inhibitory effect of these compounds, is based on studies on the recognition of RNAs by proteins in combination with in vitro RNA synthesis. For example, studies have demonstrated that the G3:U70 base pair (specifically the unpaired exocyclic 2-amino group of G3); base pairs immediately flanking the G3:U70 base pair (such as the G2:C71 base pair); and the 2xe2x80x2-hydroxyl groups of G4, C71 and U70 of tRNAAla are critical for alanine tRNA synthetase function. By taking advantage of sequence differences around these critical sites between the human tRNAAla and that of a pathogenic organism, selective drug binding can be achieved and protein synthesis by the pathogenic organism inhibited. Another example involves the interaction between the RNA-dependent reverse transcriptase of retroviruses and the specific tRNA that acts as a primer for reverse transcriptase. The annealing of the primer tRNA to the primer binding site is the first step in initiation of cDNA synthesis by reverse transcriptase, and thus represents a potential target for the arrest of viral multiplication. This can also be used as an assay for testing inhibitors of the binding reaction, for example, using glycerol gradient centrifugation to detect the presence of a complex between HIV reverse transcriptase and primer lysine tRNA.
Examples demonstrate the targeting of compounds to viral RNA which inhibit viral infection and/or replication, synthesis of compounds inhibiting viral reverse transcriptase, targeting of compounds to bacterial or pathogenic eukaryote but not human tRNA molecules to inhibit bacterial replication, and modification of compounds inhibiting rRNA to impart greater specificity and thereby decrease toxicity to normal cells.